Method of making tetrapeptide derivative TZT-1027 crystal

ABSTRACT

The invention provides crystals of N 2 -(N,N-dimethyl-L-valyl)-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl]-1-pyrrolidinyl]-1-[(S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide or salts thereof which possess potent antitumor activity and methods for their preparation.

This application is a divisional of Ser. No. 10/490,340 filed Mar. 22,2004 now U.S. Pat. No. 7,008,928, which is a U.S. national stage ofInternational Application No. PCT/JP02/09628 filed Sep. 19, 2002.

TECHNICAL FIELD

This invention relates to the novel crystals of tetrapeptide derivativewhich are useful as an active ingredient in pharmaceutical preparations,and a process for their preparation.

BACKGROUND ART

N²-(N,N-dimethyl-L-valyl)-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl]-1-pyrrolidinyl]-1-[(S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide[TZT-1027] represented by the following formula (I)

is a tetrapeptide derivative possessing potent antitumor activity, andis a potential anti-cancer agent.

TZT-1027 per se has been disclosed in, for example, PCT InternationalPublication WO93/03054 Pamphlet, which describes purification of crudeTZT-1027 with preparative thin-layer chromatography and columnchromatography, to provide amorphous TZT-1027 powder. Chem. Pharm.Bull., 43(10), 1706-1718 (1995) also describes purification of crudeproduct first by means of flash chromatography or preparative thin-layerchromatography and then column chromatography to provide TZT-1027 as anamorphous powder. JP Hei 7 (1995)-2894A, furthermore, disclosesTZT-1027, in which again crude TZT-1027 was purified by columnchromatography and preparative thin-layer chromatography to provide afluffy solid TZT-1027.

As above, amorphous TZT-1027 has been reported in the past, but noliterature disclosing crystalline TZT-1027 is found.

Generally speaking, compounds in amorphous form require more complicatedpurification operations than those in crystalline form do, andfrequently their stability is insufficient. In particular, where thecompounds are used as active ingredients of pharmaceutical compositions,their insufficient purity can provide problems. In also formulationoperations, where the compounds are in amorphous form, amorphous powderis apt to be blown up and require more cautious handling than cases offormulating crystalline compounds. For these reasons, in using TZT-1027having potent antitumor activity as an active ingredient ofpharmaceutical compositions, crystalline TZT-1027 is consideredpreferable compared to known amorphous form.

We have made various attempts to crystallize TZT-1027, but itscrystallization was very difficult because it is a peptide compound. Forinstance, TZT-1027 crystals could not be obtained throughcrystallization using such solvents as alcohols, halogenatedhydrocarbons, nitrites, ketones, organic acids, water and the like. Wefurthermore tried the crystallization using ethers such as diisopropylether, tetrahydrofuran and the like, or hydrocarbons such as n-pentane,n-hexane, cyclohexane and the like without success.

BRIEF SUMMARY OF THE INVENTION

We now discovered, surprisingly, that crystals of TZT-1027 or saltsthereof could be easily obtained by treating crude TZT-1027 or saltsthereof with a single solvent of diethyl ether or ethyl acetate, or amixed solvent formed of esters and hydrocarbons and/or ethers, and havesucceeded for the first time in the world to provide TZT-1027 or saltsthereof as crystals.

Thus, the present invention provides crystals of TZT-1027 or saltsthereof.

According to the invention, TZT-1027 or salts thereof of at least 99% inpurity, i.e., at a purity level sufficient for pharmaceuticals, throughsimple and convenient purification means such as crystallization orrecrystallization, and furthermore so obtained crystalline TZT-1027 orsalts thereof show little quality unevenness and excel in stabilitycompared with amorphous ones.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 is an IR spectrum of TZT-1027 crystal, and

FIG. 2 shows a powder X-ray diffraction pattern of TZT-1027 crystal.

DETAILED DESCRIPTION OF THE INVENTION

TZT-1027 possesses potent antitumor activity and has very strongtoxicity also to normal cells. Scrupulous care is therefore required forits purification operations and formulation operations intopharmaceutical preparations. For example, an operator's health may beendangered when he is exposed to TZT-1027 even for a short time or to aminor extent, and sufficient caution is essential for its handling. Inthe purification procedures by conventional chromatographic means orformulation procedures using the amorphous bulk, the operator's risk forbeing exposed to TZT-1027 dust is very high. Whereas, purificationprocedures of crystalline TZT-1027 provided by the present invention issimpler compared with that of amorphous TZT-1027 and causes littlescattering of dust. Formulation operations also are easier andhandlability of TZT-1027 is drastically improved.

From the result of analyzing the crystalline structure using X-rays, thecrystal system of TZT-1027 according to the present invention is foundto be orthorhombic and to have the lattice parameters of a=18.180 Å,b=24.419 Å and c=10.632 Å. Also in the powder X-ray diffraction pattern,TZT-1027 crystal provided by the present invention has characteristicpeaks at interplanar spacing (d) of 14.72, 12.27, 9.84, 9.28, 8.68,7.39, 6.11, 5.32, 5.10, 4.90, 4.61, 4.47 and 4.36 Å. Here the term“characteristic peaks” is used in the sense that they are “relativelyintense peaks” in the powder x-ray diffraction pattern, and in Table 1appearing later in this specification, the peaks having I/I₀ values of90 or higher are called “characteristic peaks”.

TZT-1027 can be crystallized in the form of a free base or, wherenecessary, first converted to pharmaceutically acceptable salts withinorganic or organic acids and then crystallized. As examples ofinorganic acid useful for forming the salt, hydrochloric acid,hydrobromic acid, nitric acid, sulfuric acid and phosphoric acid can benamed, and as examples of useful organic acid, acetic acid, propionicacid, maleic acid, fumaric acid, malonic acid, succinic acid, lacticacid, malic acid, tartaric acid, citric acid, benzoic acid andmethanesulfonic acid can be named.

As stated above, we have for the first time succeeded in obtainingTZT-1027 crystal, using as the crystallization solvent a single solventsystem of diethyl ether or ethyl acetate or a mixed solvent system ofesters and hydrocarbons and/or ethers. Whereas, TZT-1027 crystal isobtainable not only by crystallization from such solutions but also bysuch a method as crystallization from a melt.

In the present specification, “crystallization” signifies an operationto convert a compound in any form other than crystal into crystallinecompound, and “recrystallization” signifies an operation to make acrystalline compound to a compound of more purified crystalline form.

As crystallization methods from solutions, for example, concentration,gradual cooling, reaction (diffusion, electrolysis), hydrothermalgrowing and flux methods can be named. As the solvent which can be usedin those crystallization methods, where a single solvent is used,diethyl ether or ethyl acetate can be named; and where mixed solvent isused, those composed of combinations of esters and hydrocarbons and/orethers can be named. Examples of ester herein are ethyl acetate, methylacetate and the like; examples of hydrocarbon are n-hexane, n-heptane,cyclohexane, toluene, xylene and the like; and examples of ether arediethyl ether, diisopropyl ether, tetrahydrofuran, dioxane and the like.Of these, particularly such combinations as ethyl acetate/n-pentane,ethyl acetate/n-hexane and ethyl acetate/diethyl ether are suitable asthe mixed solvent. Where solvents are used in combination, the use ratiois not subject to particular limitations, while generally adequate useratios in esters/hydrocarbons or ethers are within a range of from 1/1to 1/10.

In the crystallization from solutions as above, the crystallization canbe effected by first dissolving amorphous TZT-1027 in a solvent asabove-described, e.g., diethyl ether, under heating at around 40° C.,and then cooling the solution, or first concentrating and then cooling.The TZT-1027 concentration in the solution is preferably around 15-30 wt%, and cooling to from about 0° to about 25° C. is preferred. When amixed solvent is used, the crystallization can be effected by, forexample, dissolving amorphous TZT-1027 in an ester solvent such as ethylacetate at temperatures around 40-77° C., and then adding to theresultant solution about 0.5-5 volume times the ester solvent of ahydrocarbon solvent such as n-pentane, n-hexane or the like. Here theconcentration of the amorphous TZT-1027 in the ester solvent solution ispreferably about 10-40 wt %. At the time of adding the hydrocarbonsolvent, preferably the liquid temperature is cooled to about 0-30° C.Under such preferred conditions, crystallization of TZT-1027 can becarried out with industrial advantage.

Also as crystallization methods from melts, for example, normal freezingmethod (pull method, temperature gradient method, Bridgman's method),zone melting methods (zone leveling method, float zone method), andspecial growth method (VLS method, liquid phase epitaxy method) can benamed.

Thus obtained crystals can be recrystallized, where their purity isinsufficient. Recrystallization can be carried out by repeating any ofabove-described crystallization methods or by suitably combining thosecrystallization methods.

Thus obtained TZT-1027 crystals might contain, particularly when theyare crystallized from solutions, molecules of the solvent which is usedfor the crystallization or recrystallization therein. In such a case,the solvent molecules in the crystals must be removed before thecrystals of the present invention are used as the active ingredient ofpharmaceutical compositions. Removal of the solvent can be done by, forexample, pulverizing the crystals and drying the powders under reducedpressure. Accordingly, TZT-1027 crystals of the present inventioninclude, besides those referred to as single crystal, those in a form ofcrystalline powder which is advantageously used as an active ingredientof pharmaceutical compositions.

Where crystals of TZT-1027 or salts thereof are used as an activeingredient of pharmaceutical compositions, they can be formulated intopreparation forms such as solid forms (e.g., tablets, hard capsules,soft capsules, granules, powders, fine granules, pills or troches);semi-solid forms (e.g., suppositories or ointments); or liquid forms(e.g., injections, emulsions, suspensions, elixiers, lotions or sprays),together with pharmaceutically acceptable adjuvants for drug. As usefuladjuvants in manufacturing such preparations, for example, starch,glucose, sucrose, lactose, fructose, maltose, mannitol, sorbitol,cyclodextrin, silicic acid derivatives, methyl cellulose, carboxymethylcellulose or salts thereof, alginate, gelatine, polyvinylpyrrolidone,calcium carbonate, sodium hydrogencarbonate, magnesium carbonate, talc,magnesium stearate, gum arabic, polyethylene glycol, p-hydroxybenzoicacid alkyl ester, cetyl alcohol, syrup, ethanol, propylene glycol,vaseline, carbowax, glycerine, sodium chloride, sodium sulfite, sodiumphosphate, citric acid, lactic acid, polylactic acid and polylacticacid-glycolic acid can be named.

While the content of TZT-1027 or a salt(s) thereof in such preparationsis variable depending on individual preparation forms, it is generallydesirable to use TZT-1027 or salt(s) thereof at a concentration levelwithin a range of 0.1-50 wt % for solid and semi-solid preparationforms; and at a concentration level within a range of 0.05-10 wt % forliquid preparation forms.

According to the present invention, where crystals of TZT-1027 orsalt(s) thereof are used as the active ingredient of anti-tumor agents,it is particularly preferred to use them as formulated into injectionsor orally administerable preparations.

EXAMPLES

Hereinafter the present invention is more specifically explained,referring to working examples.

In the following examples, melting points were measured with YamatoMelting Point Apparatus (MP-21 Model). Infrared absorption spectra weremeasured by the paste method according to Japanese pharmacopeia, generaltest method, with Perkin- Elmer FT-IR Spectrometer (1600 Series).Ultraviolet absorption spectra were measured with Hitachi U-3210Spectrophotometer, as to 0.01 mol/L hydrochloric acid-ethanol (95%)solution of TZT-1027, by the absorptiometric method according toJapanese pharmacopeia, general testing method. Nuclear magneticresonance spectra were measured in deuterated chloroform with JEOLJNM-LA500 FT-NMR Spectrometer, using tetramethylsilane (TMS) as theinternal standard substance. Powder X-ray diffraction was measured withMAC Science Powder X-ray Diffraction System (MXP³⁾. Optical rotation wasmeasured with JASCO DIP-140 Digital Polarimeter, as to TZT-1027'sethanol (95%) solution at a layer length of 100 mm, using sodium D ray.X-ray diffraction data of the crystals were measured with a tetraxialdiffractiometer (RIGAKU AFC7R) using Cu-Kα ray, determining the initialphase by the direct method, and the structure was made precise withSHELXL-93.

Example 1

Preparation ofN²-(N,N-dimethyl-L-valyl)-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl]-1-pyrrolidinyl]-1-[(S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide[TZT-1027] crystal:

Following the method as described at page 1718, left col., lines 20-26of Chem. Pharm. Bull., 43(10), 1706-1718, (1995), Dov-Val-Dil-Dap-OBzlwas debenzylated in t-butanol/water (9:1) in hydrogen atmosphere in thepresence of 5% palladium-on-carbon catalyst, and then reacted withβ-phenethylamine in dimethylformamide in the presence of diethylcyanophosphate and triethylamine. So obtained 5.0 g of crude TZT-1027was dissolve in 20 ml of dry diethyl ether and stirred for about 3 hoursat room temperature. Whereupon precipitated crude crystal was recoveredby filtration, to which 25 ml of ethyl acetate was added. Completelydissolving the crude crystal in the ethyl acetate at about 40-60° C.,the solution was heated and concentrated on an oil bath of about 110° C.until the distilled amount of ethyl acetate reached about 15 ml. Soconcentrated solution was gradually cooled in a water bath understirring, to about 30° C. Then, while continuing the gradual coolinguntil the bath temperature dropped to 10° C., 20 ml of n-pentane wasdripped into the solution in three divided times, followed by further 30minutes' stirring at the bath temperature of 10° C. and standing at roomtemperature for 30 minutes. The crystal whereby precipitated wasrecovered by filtration, washed twice with 9 ml of n-pentane/ethylacetate (2:1) liquid mixture, and dried under reduced pressure toprovide 4.8 g of TZT-1027 crystal (HPLC purity: 99.3%).

Melting point: 85-90° C.

¹H-NMR, 500 MHz (CDCl₃, δ): 0.81 (3H, t, J=7.5 Hz), 0.90-1.09 (3Hx5, m),1.21 (3H, d, J=7.0 Hz), 1.67-1.78 (2H, m), 1.89-1.95 (2H, m), 1.99 (1H,sextet, J=6.6 Hz), 2.07 (1H, sextet, J=6.7 Hz), 2.24 (3Hx2, s),2.35-2.40 (2H, m), 2.43 (1H, d, J=6.4 Hz), 2.83 (2H, t, J=7.0 Hz), 3.01(3H, s), 3.30 (3H, s), 3.35 (3H, s), 3.41-3.57 (2H, m), 3.84 (1H, dd,J=8.1 Hz, J=2.3 Hz), 4.05-4.08 (1H, m), 4.12 (1H, broad ddd), 4.77 (1H,dd, J=9.2 Hz, J=6.7 Hz), 6.48 (1H, broad t), 6.87 (1H, d, J=9.2 Hz),7.16-7.31 (5H, m)

IR(ν, nujol): 3330, 3250, 1640, 1621, 1090 cm⁻¹

The product's IR spectrum is shown as FIG. 1.

UVmax(HCl—C₂H₅OH): absorption maxima at 252.8 nm, 258.5 nm, 267.7 nm

[α]_(D) ²⁰: −38.4° [c=0.5, ethanol (95)]

The powder X-ray diffraction data are shown in Table 1.

TABLE 1 No. 2Theta d I(cps) I/Io FWHM  1: 6.0000 14.7167 9289 10000.1600  2: 7.2000 12.2670 2677 281 0.1800  3: 8.6800 10.1782 621 550.0600  4: 8.9800 9.8387 1726 175 0.1600  5: 9.5200 9.2825 3601 3790.2000  6: 10.1800 8.6821 1823 186 0.2200  7: 11.9600 7.3932 938 910.2400  8: 12.6200 7.0083 337 24 0.2000  9: 13.5800 6.5147 386 28 0.220010: 14.4800 6.1118 1102 106 0.2200 11: 16.6600 5.3167 1949 190 0.220012: 16.9600 5.2233 966 82 0.0600 13: 17.3600 5.1039 3010 306 0.2000 14:18.0800 4.9023 1006 90 0.2200 15: 19.2200 4.6139 1027 92 0.2400 16:19.8600 4.4667 1332 126 0.2000 17: 20.3600 4.3581 992 90 0.2400 18:22.0000 4.0369 776 68 0.3400 19: 22.4800 3.9517 396 25 0.1600 20:23.1200 3.8437 588 45 0.2400 21: 25.7000 3.4634 507 36 0.2000 22:26.4400 3.3682 523 37 0.3000 23: 27.1600 3.2805 434 28 0.2000 24:27.7600 3.2109 391 24 0.2400 25: 33.6800 2.6589 303 20 0.3000

The powder X-ray diffraction pattern is shown as FIG. 2.

Example 2

Following the method as described in Example 28 of PCT InternationalPublication WO93/03054 pamphlet, a compound obtained by deprotectingt-butyl(3R,4S,5S)-4-[N-[(N,N-dimethyl-L-valyl)-L-valyl]-N-methylamino]-3-methoxy-5-methylheptanoate(Dov-Val-Dil-Obu^(t)) in dichloromethane with trifluoroacetic acid and acompound obtained by deprotecting(2S)-2-[(1′R,2′R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl]-1-(t-butyloxycarbonylpyrrolidine)(Boc-Dap-NHCH₂CH₂-Ph) with hydrogen chloride/ethyl acetate werecondensed in dimethylformamide, in the presence of diethylcyanophosphate and triethylamine. So obtained 11.1 g of crude productwas dissolved in 48 ml of dry diethyl ether and stirred for about 3hours at room temperature. Whereby precipitated crude crystal wasrecovered by filtration, to which 20 ml of ethyl acetate was added tocompletely dissolve the crude crystal therein at about 50° C. Thesolution was gradually cooled in a water bath to about 30° C. understirring, and while further continuing the gradual cooling until thebath temperature reached 10° C., 60 ml of diethyl ether was drippedthereinto in three divided times. After the dripping, stirring wascontinued for further 30 minutes at the bath temperature of 10° C.,followed by 30 minutes' standing at room temperature. The precipitatedcrystal was recovered by filtration, washed twice with 20 ml of diethylether/ethyl acetate (3:1) liquid mixture, and dried under reducedpressure to provide 8.42 g of TZT-1027 crystal (HPLC purity: 99.2%).

Example 3

Preparation of a crystal for analyzing the single crystal structure:

Two (2.0) mg of crystalline powder of the TZT-1027 which was obtained inExample 1 was dissolved in 0.5 ml of ethyl acetate, and 0.5 ml ofn-pentane was added to the formed solution. The sample tube containingthis solution was put in a vessel containing 5 ml of n-pentane andsealed hermetically. Allowing the sealed vessel to stand in arefrigerator maintained at about 0° C. for 5 days, a crystal suitablefor analyzing the single crystal structure was obtained.

The crystalline data obtained by the structural analysis of the singlecrystal are as shown in the following Table 2.

TABLE 2 Compositional formula: C₃₉H₆₇N₅O₆ Molecular weight: 701.98Crystal color: colorless Crystal configuration: prismatic Crystalsystem: orthorhombic system Space group: P2₁2₁2₁ Lattice constants: a =18.180Å b = 24.419Å c = 10.632Å Unit lattice volume: V = 4719Å³ Numberof molecules in unit lattice: Z = 4 Crystal density (calculated): Dcalc= 1.089 g/cm³ R factor: R(F) = 0.050 (I > 2σ(I))

1. A method of making crystals of N²-(N,N-dimethyl-L-valyl)-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl]-1-pyrrolidinyl]-1-[(S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide(TZT-1027) represented by the following formula (I) or a salt thereof:

which comprises crystallizing amorphous TZT-1027 or a salt thereof fromdiethyl ether; and optionally recrystallizing the formed crystals. 2.The method according to claim 1, in which the recrystallization isperformed from a mixed solvent selected from combinations of esters andhydrocarbons and/or ethers.
 3. The method according to claim 2, in whichthe ester is selected from the group consisting of ethyl acetate andmethyl acetate; the hydrocarbon is selected from the group consisting ofn-pentane, n-hexane, n-heptane, cyclohexane, toluene and xylene; and theether is selected from the group consisting of diethyl ether,diisopropyl ether, tetrahydrofuran and dioxane.
 4. The method accordingto claim 2, in which the ratio of ester/hydrocarbon or ether is within arange of from 1/1 to 1/10.
 5. The method according to claim 2, in whichthe ester is ethyl acetate; the hydrocarbon is n-pentane or n-hexane;and the ether is diethyl ether.
 6. The method according to claim 2,which comprises dissolving TZT-1027 crystals in an ester solvent at atemperature around 40-77° C., and then adding to the resultant solutiona hydrocarbon or ether solvent in about 0.5-5 volume times the amount ofthe ester solvent, optionally after cooling the solution to a liquidtemperature of about 0-30° C.